Animal feed containing polypeptides

ABSTRACT

The present invention relates to an animal feed made of an amount of cereal grain and a peptide or polypeptide expressed by a transformed organism, whereby the transformed organism can be included in the animal feed composition. The present invention also relates to a method for forming the animal feed wherein the method includes forming a transformed organism by transforming a yeast cell by inserting a nucleic acid molecule which will express a polypeptide desired for use in the animal feed.

RELATED APPLICATIONS

This application is a divisional of commonly-owned, U.S. applicationSer. No. 09/613,666, filed 11 Jul. 2000 now U.S. Pat. No. 6,737,262.

SEQUENCE LISTING

This application is accompanied by a Sequence Listing for nucleotidesequences.

FIELD OF INVENTION

The present invention relates to an animal feed comprised of an amountof cereal grain and an amount of polypeptide, with such polypeptideexpressed by a transformed organism. More particularly, the presentinvention relates to a genetically engineered yeast cell that expressesa polypeptide that is comprised of amino acids necessary to animalnutrition and growth, and methods related thereto.

BACKGROUND OF INVENTION

Cereal grains, such as corn, wheat, and rice, are fed to livestock andother animals, with consumption of these grains causing the animals togain weight. Generally, consumption of such grains and/or grass resultsin sufficient weight gain, however, it is preferred to produce animalswhich have enhanced weight gain, as this increases the value of theanimal. In other words, it is desired to produce animals of exceptionalsize and weight, which are comparatively larger than animals fed anormal diet. Increased weight gain can be promoted by ensuring that theanimal receives necessary amino acids in desired amounts which helppromote weight gain and are important nutritional constituents. Itshould also be noted that many amino acids which help enhance weightgain in various animals are not readily found in grass and cereal grainstypically consumed by such animals. As such, farmers, ranchers, and feedlots supplement the animal's diet with various compositions whichcontain many of the desired amino acids.

One way to ensure that an animal's diet is supplemented with amino acidsin amounts sufficient to result in weight gain, is to add amino acidsderived from various natural sources to the animals' feed. Natural aminoacid sources include offal from the slaughter of animals for humanconsumption, slaughter house sludge, poultry byproduct meal, orlivestock or poultry litter. An example of how this material is used topromote growth can be illustrated by the use of the sludge. The sludge,which includes blood and feces from the floor of a slaughter house isgathered, treated, and mixed with animal feed to provide an animal withamino acids. Often, bacteria, such as E. coli, Salmonella, or otherpathogens, are residents of such sludge and waste. If the sludge orwaste is not thoroughly treated then infectious agents can betransmitted to animals that are fed the amino acid enhancements. Therisk of transmitting infectious agents to animals is a growing concernas witnessed in Europe by the reaction to “mad cows' disease.” Thus, itis greatly desired to have a method for supplying enhanced levels ofamino acids to animals that does not require the use of amino acidsderived from animal waste or sludge.

An alternative to using animal waste products is to produce thenecessary and desired amino acids through genetic modification ofvarious microorganisms. Currently, most amino acids used to supplementanimal feed are synthesized by microbial fermentations. Such amino acidsare then purified and can be added to animal feed as a supplement. Mostof these amino acids form heterologous peptides. The use of thesetechniques has been undesired because the cost has made the addition ofsuch amino acids prohibitive. Another problem is that the excreted aminoacids must be purified. Most purification methods include stabilizingthe amino acid using hydrochloride with lysine. This converts D isomersto L isomers. Consequently, it is further desired to have a method orcomposition for adding amino acids to animal feed that does not requirea purification step and is economical and cost effective.

Purified amino acids produced by genetically modified organisms,typically bacteria, suffer from a number of other problems in additionto the cost. Often, purified amino acids are subject to degradativereactions during processing, such as maillard reactions. As such, it ishypothesized that providing the amino acids in a protein or polypeptideform would prevent or render such amino acids less susceptible to suchdegradative reactions. Often, pure amino acids are susceptible toincongruous absorption and catabolism by the intestinal or hepatictissue of the animal consuming such products. For this reason, it isdesired to have an amino acid or protein peptide that allows for easydigestion and absorption to be preferably accomplished congruently withother dietary proteins.

Finally, when using purified amino acids, only a limited number can beprovided to the animal. As such, it is desired to have a method orcomposition that allows for a protein or polypeptide to provide secondand third limiting amino acids.

The use of yeast and other microbes to express polypeptides is known.For example, U.S. Pat. No. 5,856,123 ('123) discloses a DNA expressionvector for expressing a polypeptide. The vector includes bacterial andyeast origins of replication and genes for phenotypic selection of bothbacterial and yeast moieties. The expression vector is designed forexpressing A or B chains of human insulin. The patent does not disclosethe use of a transformed yeast cell to produce the amino acids necessaryto animal nutrition. Nowhere is an animal feed mentioned in the '123patent. While this particular patent is indicative of a variety ofdisclosed methods and compositions which discuss the use of transformedyeast cells for producing polypeptides or proteins, it is believed thatthe use of transformed yeast cells to produce animal feeds has not beendisclosed. It is believed that the use of transformed organisms toproduce polypeptides comprised of different amino acids desired inanimal nutrition has not been practiced.

Yeast has also been known to be used to produce lactic acid. Metabolicengineering has focused on the production of heterologous proteins andpeptides, including producing such proteins or peptides in transformedyeast. It is desired, however, to produce polypeptides comprised of morethan one amino acid residue.

For these reasons, it is desired to have a method for producing ananimal feed that is economical and does not run the risk of microbialcontamination. Additionally, it is desired to have an easily activatedvehicle that readily delivers amino acids for animal consumption. Mostimportantly, it is desired to have an animal feed having desired typesand concentrations of amino acids.

SUMMARY OF INVENTION

The present invention relates to an animal feed that includes an amountof cereal grain and a peptide or a polypeptide expressed by atransformed organism. The polypeptides can be separated from thetransformed organism, or the transformed organism and polypeptide,together, can be added to the cereal grain. The present inventionfurther relates to the transformed organism which is preferably a yeastcell having a nucleic acid polymer inserted into the yeast cellchromosome, whereby the nucleic acid molecule can be expressed to form apeptide or polypeptide useful in the nutrition of an animal. The nucleicacid polymer will preferably be comprised of a fragment for expressingthe polypeptide, a promoter that can be induced, and an identifier whichis preferably either histidine or uracil related. The promoter ispreferably a GAP promoter. While yeast is the preferred organism to betransformed, any of a variety of other microorganisms that can beconsumed by animals and which can be transformed may also be used.

The present invention also relates to a method for forming thetransformed organism and a method for forming the animal feed comprisedof the cereal grain and either the transformed organism or theexpression product of the transformed organism. The method includespreferably forming a synthetic nucleic acid polymer and attaching apromoter and stop sequence thereto. After formation of the nucleic acidpolymer, the method will further include inserting the polymer into avector and transforming a host organism. Alternatively, the integrationconstruct can be placed in the host organism by using electro-poration.It is preferred if prior to transformation, the nucleic acid polymer isannealed or attached to primers which will allow for the formation of anintegration construct. The resultant integration construct is thenplaced in a transfer vector and used to transform the host cellorganism. It should also be noted that it is preferred if thetransformed yeast cell is haploid, because it is preferred to insert thenucleic acid polymer into the chromosome of the host. Diploid hosts canbe used if the nucleic acid polymer is inserted outside the chromosome.

The polypeptide or peptide expressed by the transformed organism will beat least two amino acids long, and will be comprised of at least twodifferent amino acid residues. More preferably, the polypeptide will bebetween 20 and 30 amino acids long and will be comprised of at leastfive different residues. The amino acids to be expressed by thetransformed organism will be selected in advance to ensure that, whenconsumed, the animal will receive maximum nutritional benefits so as toenhance growth and weight gain. Such amino acids can be selectedaccording to the animal's age and particular nutritional needs.

The present invention is advantageous because it allows for aneconomical way to produce amino acids, specifically polypeptides, thatcan then be used to supplement an animal's diet. The present inventionis especially advantageous because the transformed host organism canalso be fed to the animal. This means purification steps can beeliminated so that costs are minimized. The invention is furtheradvantageous because the amino acids are readily absorbed and do notreadily degrade. Also, the polypeptide will include amino acid residuesnot typically found in other animal feeds or supplements. Thus, ananimal feed is produced that is more nutritionally complete because itcontains desired amino acids believed not to be found in other animalfeeds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates PCR construction of a synthetic gene for encoding asynthetic peptide, whereby a 68 by primer dimer is formed;

FIG. 2 shows the formation of a full length synthetic gene using the 68by primer dimer as a template;

FIG. 3 shows the insertion of the synthetic peptide gene into a plasmid,having a stop sequence, a marker, and a promoter;

FIG. 4 illustrates a 698 by synthetic gene product having a syntheticpeptide and a promoter;

FIG. 5 shows the synthetic gene of FIG. 4 being blunt end cloned into aplasmid yeast shuttle vector pRS 308;

FIG. 6 shows an integration construct to be inserted into an HO geneside of a haploid yeast strain YJZ001a and YJZ001b;

FIG. 7 shows the integration construct and portion of resultanttransformed organism;

FIG. 8 shows an exemplary construct of various primers used to form theconstruct of the present invention; and,

FIG. 9 shows an exemplary integration construct of the presentinvention.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to an animal feed comprised of an amountof cereal grain and an amino acid polymer, with it more preferred forthe animal feed to be comprised of a host for expressing the polymer.More preferred is for the animal feed to be comprised of cereal grainand at least one additive peptide, polypeptide, or protein. The presentinvention also relates to a method for forming the animal feed. Includedin the method for forming the animal feed is a method for introducing anucleic acid molecule or polymer into a host organism, with expressionthen occurring in the host organism to form the peptide or peptides. Itis preferred if a transformed yeast cell is formed for expressing thepeptide. Another aspect of the invention relates to a vector forintroducing the nucleic acid molecule into the host organism, with thevector having a synthetic gene for expressing a peptide or, at the veryleast, containing a nucleic acid molecule that can be expressed to forma desired peptide. Preferably, a synthetic gene or genetic constructcomprised of the nucleic acid molecule and a promoter can be insertedinto the vector, with the gene designed to express a specific group ofamino acids. Further, the present invention relates to a gene, and moreparticularly a nucleic acid polymer, that when expressed will produce adesired peptide designed to maximize weight gain and growth in ananimal. It is preferred if the animal feed is comprised of an amount ofthe grain and an amount of the peptide, whereby the peptide is producedby a transformed yeast cell, with a vector or near DNA fragmentcontaining a synthetic nucleic acid polymer used to transform the yeastcell that can then be expressed to form the peptide.

The animal feed, as mentioned, is comprised of an amount of grain and anamount of a product expressed by a microorganism, preferably an aminoacid polymer. Preferably, the amino acid polymer is a peptide orpolypeptide, although proteins may also be useful in the present animalfeed. The feed will contain at least one peptide, but may containmultiple separate peptides, with each separate peptide comprised ofdifferent amino acids. The animal feed is designed to provide animalswith necessary amino acids so that growth and health can be enhancedwhen the animal consumes the present feed on a regular basis.

The feed is intended for consumption by livestock, companion pets,aquacultures, and captive animals, with livestock including poultry,bovine, ovine, porcine, and equine members. Captive animals will includethose that are maintained in zoological parks or hunting or conservationreserves. Companion pets include felines and canines, and aquaculture isdefined to include all species of fish or invertebrates used in foodproduction for which a soy-based diet is being used or may be used withnutritional adjustment. Essentially, any animal that can be fed on aconsistent basis grain based animal feed having an amount and desiredconcentration ratio of amino acids, preferably peptides, can be fed thepresent inventive feed. All of these different types of animals willconsume different types of cereal grains and have different amino acidrequirements. Thus, according to the present method, animal feedsdesigned to meet the nutritional requirements of individual species canbe manufactured by expressing nucleic acid polymers to produce desiredpolypeptides, with the nucleic acid polymers being either synthetic,isolated and naturally occurring, or combinations thereof.

The cereal grains which can be used to form the feed of the presentinvention include, but are not limited to, soybean, corn, barley, rice,wheat, oats, millet, maize, sunflower, canola, grass, and combinationsthereof. The expression products, preferably a peptide or peptides, thatare mixed with the cereal grains to form the present feed can bespecified so that dependent upon the particular animal to which theanimal feed is fed, necessary amino acid constituents can be specified.More particularly, amino acids necessary for weight gain and generalhealth can be specified, so that the feed is animal specific. Further,the feed, in particular the peptides, can be adjusted so that dependentupon the age or development of the animal, particular types andconcentrations of amino acids can be included in the animal's diet.

Any expression product that promotes increased weight gain and betterhealth in the animal being fed can be mixed with the grain. Thepreferred expression product is a peptide or polypeptide, with thepeptide preferably exogenous to the host organism. A peptide is definedherein to be comprised of at least two amino acids attached to oneanother via a peptide bond or other covalent bond, with the polypeptidestypically comprised of between about 10 and about 30 amino acids. Theamino acids are defined as containing an α-amino group (NH₂) and anacidic α-carboxyl group (COOH), with it known that there are 20identified naturally occurring amino acids in eukaryotic proteins, alongwith L and D isomers. The available amino acids include alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. As such, the peptides are strings of connected amino acids. Theamino acids which form the peptide can be all the same or combinationsof different L or D amino acids.

The first step necessary to forming the animal feed and providing aparticular animal with desired expression products involves selecting adesired cereal grain. The cereal grain selected is dependent upon theparticular dietary requirements of the animal to be fed. After thecereal grain is selected, the particular expression product, typically apolypeptide, necessary to promote increased weight gain must bedetermined. This generally requires identifying desired and particularamino acids and the concentration and ratio of such amino acids. Oncedetermined, a peptide or polypeptide comprised of these amino acids mustbe formed. Such amino acid information is available in standardveterinary and animal science protocols, and can be used to eitherselect a naturally occurring nucleic acid polymer, or to form asynthetic nucleic acid polymer. For example, the following table showsthe ideal amino acid requirements for three different species of adultanimals.

Poultry Swine Dairy Beef Lysine 100 100 100 Methionine/Cysteine 60 33 20Threonine 60 0 0 Valine 75 0 0 Isoleucine 60 0 0 Arginine 80 0 100Tryptophan 20 0 0 Histidine 0 0 35

Note that amino acid requirements for all animals are determined in viewof providing the animal with 100% of the recommended amount of lysine.Thus, the listed amino acids are the desired weight percent added ascompared to lysine.

As can be seen, an adult chicken requires lysine, threonine, methionine,etc. A poultry feed preferably contains these percentages of amino acidsin amounts sufficient to ensure all consuming subjects will receive thedesired amount of specified amino acids. The amino acids are deliveredas peptides, polypeptides, or proteins. A preferred poultry feed will becomprised of ground corn or soybean meal and a polypeptide, with thepolypeptide preferably comprised of the following amino acid units: 3methionine, 6 histidine, 6 lysine, 2 threonine, 2 isoleucine, 1 valine,and 1 tryptophan residue. Other acceptable polypeptides for use with theanimal feed are listed in Example 9.

To have a desired peptide expressed, it is necessary to have a nucleicacid polymer, also known herein as a gene, for expressing such peptide.Acquisition of such gene is accomplished, for example, by isolating agene or a host organism that expresses the gene, isolating a nucleicacid molecule or polymer which encodes for a desired peptide and forminga synthetic gene based on this molecule or polymer, forming a syntheticnucleic acid molecule or polymer which encodes for a desired peptide andforming a synthetic gene, or isolating a nucleic acid molecule orpolymer ligating a synthetic nucleic acid molecule or polymer theretoand forming a synthetic gene. Dependent upon the peptide, which willcontain the desired amino acids, concentrations, and ratios thereof,either a nucleic acid molecule or polymer that encodes the peptide isisolated, a nucleic acid molecule or polymer is formed, or a syntheticnucleic acid molecule or polymer is attached to an isolated nucleic acidmolecule. Because the specific concentration and combination of aminoacids often is not expressed by a naturally occurring nucleic acidmolecule, it is generally necessary to form a synthetic nucleic acidmolecule or polymer. Keep in mind, any of a variety of nucleic acidmolecules may express the same desired peptide. The amino acids whichcomprise the peptide can be arranged in any order, meaning the codonsfor the amino acid can be arranged in a variety of orders in the nucleicacid molecule or polymer. Note that a codon of 3 nucleic acid basesmight be considered a molecule of a nucleic acid, but several codons isgenerally considered a nucleic acid polymer.

If a nucleic acid polymer that expresses the desired peptide isavailable, standard protocols for isolating such molecule are followed,including removing the polymer from the host genome. This method willinclude using an enzyme (restriction endonuclease) to cut the nucleicacid fragment out of the host genome or episomal DNA molecule. Moreparticularly, the fragment is identified and a restriction digestion isconducted to isolate the fragment. Any of a variety of restrictionenzymes can be used as long as the fragment is cut and isolated from thehost. An example of a suitable protocol was published in Birren et al.(1997).^(1, 2, 3)

Note that if a host organism exists that is non-toxic, easily grown, canbe fed to animals, and contains an easily expressed nucleic acid polymerthat expresses a desired peptide, then transformation is not necessary.In this case, the polymer merely needs to be expressed and the productmixed with the feed. Further, the transformed organism can be inducibleor constitutive dependent upon what is desired. Typically, the isolationof such organism is unlikely because of the necessity of expressing awide range of peptides and polypeptides; however, if such an organismexists, it can be used in the present invention.

Conversely, if the nucleic acid molecule or fragment is not naturallyoccurring, it is necessary to make a synthetic fragment. Typically, itis necessary to form the synthetic fragment, as the preferred type andconcentration of amino acids are not available as a result of expressionof the nucleic acid molecule or polymer. It is generally necessary andpreferred to make a fragment that, when expressed, results in a desiredpeptide or polypeptide. A standard protocol for forming such fragment isfound in Birren et al. (1997).^(1, 2, 3) Once the fragment is formed orisolated, it is necessary to transfer the fragment to a host organism.This may require a number of steps. It is most preferred to form asynthetic fragment, but isolated nucleic acid molecules or polymers andhost organisms can be used.

The synthetic fragment for expressing the peptide preferably iscomprised of two single stranded fragments annealed to one another, butfragments that are initially double stranded may also be used. The mostpreferred method for forming the fragment is initiated by acquiringsingle stranded primers which will anneal to one another to form adouble stranded fragment. The primers are comprised of nucleic acids,more specifically codons, which express the desired peptide. The primersare formed according to the desired specification related to theexpressed peptide. The protocol for forming a primer comprised of thedesired nucleic acids is detailed in Gait 31. Additionally, the primershave recognition sites whereby an endonuclease recognizes and cuts themolecule at the specific site. One primer should be a 3′ to 5′ strandhaving a known nuclease site; for example, a 3′ fragment having an Not Icut site. The other primer should be a 5′ to 3′ fragment having a knownnuclease site. The primers are combined and then passed through a PCRprocess to form a primer dimer. Increased amounts of product will beformed as a result of the primers being subjected to the PCR process.The primers will anneal to one another to form the primer dimer. Theprimer dimer will contain nuclease recognition sites on each end andcodons for expressing the desired peptide. Also, the primer dimer willpreferably have blunt ends.

If the primers do not anneal, it may be necessary to use a patch topromote attachment of the primers. The patch, like the primers, will becomprised of nucleic acids designed to express a desired peptide.Attachment results from first annealing the patch to a primer andpassing the product through a PCR step. The patch primer product is thenannealed to another primer and again passed through the PCR. Anillustration of this involves a primer patch and a primer coding forpart of the peptide annealing the two and amplifying to form a 68 basepair (bp) primer dimer. The 68 by primer dimer is then combined with a5′ Synpep Eco R1 primer and passed through the PCR process to form a 91by synthetic peptide fragment having an Eco R1 site and a Not I site.Additionally, contained in the fragment is a Kozack consensus fortranslation start.

While the preferred method for forming a synthetic nucleic acid moleculeinvolves annealing two primers containing the desired codons, othermethods may be used. In fact, any method can be used that will result ina nucleic acid molecule or fragment that expresses the desired peptide.

PCR (Polymerase Chain Reaction) is a system used for DNA replicationthat employs the essential enzyme of cellular DNA replication, DNApolymerase. The primers used to form the primer dimer bracket thenucleic acid molecule that expresses the peptide and serve as points ofattachment for the polymerase. Amplification occurs so that a sufficientamount of material is available to continue the transformationprocedure. The procedure for a typical PCR reaction can be found inInnis et al. (1990)¹¹.

Once the fragment is formed or isolated, it is necessary to attach apromoter to the fragment, if one is not present, so that the nucleicacid polymer can be turned on or transcribed and expression of thepolypeptides will occur. A different, yet equally acceptable approachinvolves inserting the synthesized fragment into a naturally occurringnucleic acid polymer in such a way that the fragment is controlled bythe polymer's promoter sequence. The promoter is the region of a nucleicacid polymer required for maximum expression of the gene. The promotercan be isolated or can be synthesized, based on the known sequence of anexpressed gene of prokaryotic or eukaryotic origin.

Promoters can be induced or constitutive. An induced promoter means itcan be turned on in response to a constituent that induces activation.Conversely, a constitutive promoter is not turned on or off, butcontinues to cause expression of the fragment. An example of aconstitutive promoter is where the gene construct contains atranscription elongation promoter (TEF). Either type of promoter isacceptable as long as suitable expression of the polypeptide occurs. Inthe present invention, a GAPDH (glyceraldehyde-3-phosphate dehydrogenasepromoter) promoter of 679 nucleic acid base pairs is most preferred.This is a constitutive promoter.

Another preferred method for expressing the peptide or polypeptiderelates to adding Cu⁺ to the medium in which the host organism,transfected with the fragment, is grown. This is a copper promoterinduction system. Alternatively, promoters induced by alcohol,glycolytic pathway, and carbon source can be selected.

It is preferred if the promoter is isolated as opposed to synthetic. Anyof a variety of promoters can be selected, with it preferred that thepromoter selected be derived from the organism that is to betransformed. For example, if a yeast cell is to be transformed toexpress the polypeptide, then it is preferred if the promoter is yeastderived. Promoter sequences which can be used in the present invention,include, but are not limited to: AOX1 (alcohol oxidase), which isinducible by methanol and is repressed when glucose or galactose is usedas a carbon source; GAP (glyceraldehyde-3-phosphate dehydrogenase)providing a strong constitutive expression when the yeast is grown onglucose; FLD1 (glutathione-dependent formaldehyde dehygrogenase), a keyenzyme when methylated amines are the nitrogen source and methanol, thecarbon source for methyloptrophic yeasts; PEX 8 gene (peroxisomal matrixprotein); and the YPT1 gene (encodes GTPase involved in secretion). TheGAP promoter is one of the most commonly used in S. cerevisiae as well,but is of little use if the protein produced is toxic to the yeast. Alist of suitable promoters is as follows:

Examples of promoters designed to obtain heterologous protein expressionin yeasts. Yeast species indicated. Sc=Saccharomyces cerevisiae;YI=Yarrowia lipolytica, So=Schwanniomyces occidentalis, Ps=Pichisstipitis, Pp=Pichia pastoris, Hp=Hansenula polymorpha, KI=Kluyveromyceslactis

Promoter Yeast* Reference CYC1 Sc Cantwell et al., 1986⁴ ADH1 ScCantwell et al., 1986⁴ GAL7 Sc Buckholz & Gleeson, 1991⁵ ADH2 ScSchuster, 1989⁶ GAPDH Sc Schuster, 1989⁶ LEU2 Y1 Davidow et al., 1990⁸XPR2 Y1 Davidow et al., 1990⁸ TEF Y1 Mueller et al., 1998⁹ RPS7 Y1Mueller et al., 1998⁹ URA3 Y1 Davidow & DeZeeuw, 1991⁷ CUP1 Sc Hinnen etal., 1994¹⁰ ENO Sc Innis et al., 1985¹¹ GAL1/GAL10 Sc Schulte et al.,1987¹² GAPDH Sc Hallewell et al., 1987¹³ PGK Sc Kingsman et al., 1987¹⁴PHO5 Sc Hori et al., 1990¹⁵ Mfool Sc Brake et al., 1984¹⁶ GAM1 So/PsPiontek et al., 1998¹⁷ XYL1 So/Ps Piontek et al., 1998¹⁷ ADH1 So/PsPiontek et al., 1998¹⁷ PDC1 So/Ps Piontek et al., 1998¹⁷ AOX1_(p) PpOgawa et al., 1999¹⁸ Hp Raschke et al., 1996¹⁹ MOX_(p) Hp Raschke etal., 1996¹⁹ FMD Hp Gellissen & Hollenberg, 1997²⁰ GAP Pp Waterham etal., 1997²¹ FLD1 Pp Shen et al., 1998²² PEX8 Pp Johnson et al., 1999²³YPT1 Pp Sears et al., 1998²⁴ LAC4 K1 Van den Berg et al., 1990²⁵ PGK K1Rocha et al., 1996²⁶ Sc Rocha et al., 1996²⁶ CUP1 K1 Gellissen &Hollenberg, 1997²⁰ Mfool K1/Sc/Hp Gellissen & Hollenberg, 1997²⁰ CTTI K1Gellissen & Hollenberg, 1997²⁰ ADH4 K1 Saliola et al., 1999²⁷ AMY1 SoPiontek et al., 1998¹⁷

It is also necessary to attach a transcription stop sequence to thefragment. This is done so as to stop transcription of the DNA fragment.Preferably, the transcription stop sequence is also derived from thehost organism. Attachment typically occurs when the promoter isattached.

Any of a variety of methods can be used to attach a promoter to form thesynthetic nucleic acid polymer. The preferred way to attach a promoterand a stop sequence to the synthetic peptide fragment is to clone thefragment for expressing the peptide into a plasmid containing thedesired promoter and stop sequence. This is done by selecting a plasmidhaving available restriction nuclease sites the same as the syntheticfragment with the plasmid then cut with endonucleases and the fragmentthen cloned into the plasmid at the cut site. For example, as statedabove, the preferred synthetic fragment has an Eco R1 site and a Not Isite. Thus, a plasmid having a suitable promoter and stop sequence, andEco R1 and Not I sites can be selected with the plasmid cut by therestriction endonucleases and the fragment cloned therein. It ispreferred if the fragment is located proximal to the promoter in theplasmid. Any cut site can be used, with the sites designated, based onthe plasmid of choice. There are well over 400 nucleases that can beused. It is also preferred if the plasmid includes an identifier. Suchidentifiers include:

MBP maltose binding protein His(6) polyhistidine MYC transcriptionfactor GST glutathione S transferase

The promoter and synthetic peptide region that create the syntheticnucleic acid polymer will preferably contain a region that allows fortesting of expression of the gene in a host organism. The identifier isdesired because it allows for confirmation as to whether an organism hasbeen transformed. An example of a suitable identifier is an myc epitope.This is a histidine site in the synthetic gene which means that thesynthetic gene will express histidine. Thus, when a host is transformed,the transformation can be checked for by using a western blot thatdetects antibodies, such as polyhistidine. Other examples includemarkers inducing auxotrophy or antibiotic resistance.

Combining the promoter with the fragment and marker includes threedifferent approaches:

-   -   (a) a zeocin selectable integrant;    -   (b) a URA 3 selectable integrant, which uses an auxotrophic        marker such as HIS, Trp, LEU 1, or LEU 2; and,    -   (c) URA 3 selectable plasmid.

The preferred nucleic acid polymer has a GAPDH promoter, URA3 marker, ayeast stop sequence, and a nucleic acid polymer coding for a desiredpeptide or polypeptide. The expressed polypeptide is comprised of 3methionine, 6 histidine, 6 lysine, 2 threonine, 2 isoleucine, 1 valine,and 1 tryptophan residues.

While a plasmid containing a stop sequence, promoter, and identifier ispreferred, other methods can be used to attach the promoter to thenucleic acid molecule or polymer. An alternative method would includeattaching a primer to the nucleic acid molecule, with the primercomprised of the promoter necessary to transcribe the nucleic acidmolecule. Other methods may also be used as long as the promoter isattached to the fragment.

Once it has been determined that the synthetic nucleic acid polymer canbe formed by cloning the fragment into a plasmid, it is necessary toamplify the synthetic polymer so as to have enough product to transformthe desired host organism. The synthetic polymer or gene, comprised ofthe promoter, the nucleic acid polymer for expressing the peptide, themarker, and the stop sequence, is cut out of the plasmid. This isaccomplished by again using a method involving different availablerestriction endonucleases. The gene fragment is then amplified usingPCR. This is the preferred way to amplify the synthetic gene.

Once the synthetic gene is formed and in a sufficient amount, it isnecessary to insert the gene into a host organism for expression. Any ofa variety of means can be used to transform the host. It is mostpreferred to use a shuttle vector to transport the gene or nucleic acidpolymer to the host and transform such host. Any of a variety of vectorsare available for use in transferring the construct, including plasmids,cosmids, phagemids, and artificial chromosomes.

The synthetic gene can be cloned into a vector which can be used totransform the desired host organism. It is most preferred to use aplasmid yeast shuttle vector to transform the host yeast cells. Any of anumber of plasmids can be used as long as the gene is transferred to thehost, such host is transformed, and the gene can be expressed. In thepresent case, it has been observed that the synthetic gene can beblunt-end cloned into a yeast shuttle vector, such as a pRS 316 or 308having Eco RV and Not I sites. These plasmids are preferred because theycan confer an auxotrophic mutation. The plasmid vector is cut with anuclease, and the synthetic gene is then blunt-end cloned into thevector to form a transformed vector. More specifically, it is preferredto double cut the plasmid with two nucleases to form a cut plasmid. Oncethe plasmid is cut by the enzymes, which typically occurs at 37° C. fortwo hours, the plasmid and gene are then ligated in the presence of theT4 ligase enzyme by melting the constituents for a period of time at adesired temperature, such as five minutes at 42° C. and then cooling to16° C. for at least one hour. The plasmid product is purified andquantified, with a genetic vector construct formed.

In the case of yeast, a variety of 1.6-2.0 μm plasmid vectors are alsoavailable, which are epigenetic. Other suitable vectors insert thenucleic acid polymer into the nucleus or chromosome or ribosomalchromosomes. It is also preferred if the vector is a hybrid of abacterial and yeast plasmid. Included in such a hybrid should be asequence for bacterial and yeast replication. Preferably, the plasmidwill contain ARS element vectors, which are designed to replicateindependent of the genome and can increase to multiple copies oftenexceeding 20 per cell. Early ARS vectors were unstable and could bestabilized by addition of a yeast centromeric sequence (Romanos et al.,1992)²⁸. More recently designed ARS vectors, such as those of the pINAtype (Fournier et al., 1993)²⁹ and the pGC type (Müeller et al., 1998)⁹are relatively stable and are being used as high-copy number ofexpression vectors. Suitable plasmid vectors include, but are notlimited to, pHIL-D2, pA0815, pPIC3K, pPICZ, pHW010, pGAPZ, pHIL-S1,pGApZ∞, pPIC9K, pPICZ∞, pIXY654, PDK1, pRBGO, pA0816, pA0817, pHWO18,PSS050, pSSO₄₀, pAR0815∞, pAR0815PDI, pHIL-D2pro1(III), pMP8, pMIVIST,pMIVINS, YEpB2plys.DELTA49, YEpZ100, YepZ101, YepZ415, pG12062, pGG5,pGG53, pGP3 Hr, pGC69-3, pGC69-4, pXC69-4, pXX33, pXX22, pSP24, pLS-3,pC5aX3, pLD56, pLX-34, pPIC3X, pTL2M, pAS7/pAU5, and pSL2P3M.

After the vector construct is formed, it is necessary to transform thehost organism with the vector. Any host that is suitable for use in ananimal can be transformed. The host organism can be any of a variety ofnon-toxic eukaryotes, including fungi, or prokaryotes. Moreparticularly, the host can be anything that can be consumed by theanimal being fed. Most preferably, the host organism will be a yeastcell with available host yeast cells selected from the following genera:Saccharomyces cerevisiae, Pichia spp (including P. pastoris and P.stipidis) Yarrowia spp (including Yarrowia lipolytica), Candida spp,Kluyveromyces ssp (including K. waltii, K. lactis and K.drosophiliarium), Zygosaccharomyces spp, Schwannomyces occidentalis,Schizosaccharmyces pombe, Hansenula spp (including H. polymorpha) andTorulaspora delbrueckii. The plasmids or vectors and host cells areincubated at a sufficient temperature and time to cause the vector totransfect the host cell.

Before producing the finished host product, it is preferred to test thehost for expression of the gene. This is similar to testing the host toensure transfection has occurred. An example of a useful testing methodincludes transforming a non-auxotrophic yeast strain. The construct, asmentioned, is auxotrophic (-URA), so that the transformation istheoretically easy to accomplish and an auxotrophic mutant is formed.The auxotrophic mutants are typically used as a testing system becausethis is considered comparatively easy. This results in an easyidentification system which, in turn, permits easy determination ofwhether the host was transformed. Another example is to test forantibiotic resistance, which will be imparted to the host by theconstruct. In addition to the auxotrophic markers, a western blot orother techniques can be used to test for expression. For example,histidine should be expressed if the yeast cells have been transformedwith a synthetic gene that codes for histidine. The western blot willuse antibodies (polyhistidine (His)₆) to detect the presence ofhistidine. If transformation has occurred, as demonstrated byexpression, it is then necessary to create an integration construct forinsertion of the synthetic gene into yeast strains or other hostorganisms which will be used to express the desired peptide.

As mentioned, the integration construct is formed by isolating a linearconstruct from the cloned vector, whereby the construct will becomprised of the promoter, nucleic acid polymer for expression of thepeptide, and part of the vector plasmid. Preferably, the auxotrophic(URA) marker or other marker gene will be included in the construct. Inorder to transfect the host, the following discussed steps arepreferred. The construct is isolated by cutting the plasmid with thenecessary nuclease and then amplifying with PCR. Next, the construct isprepared for insertion into the host. A gene site is identified in thehost where the synthetic gene can be inserted. Primers are attached tothe ends of the integration construct. The primers will form a homologyregion which will allow for the exchange of a chromosomal gene in thehost for the synthetic gene. The homology region will be comprised of anumber of non-transcribed base pairs. Generally, 40 base pairs should behomologous between the host chromosome and the integration construct,plus or minus 10 base pairs. Also, the primers that form the homologyregion will be 5′ to 3′ and 3′ to 5′. The primers will be homologous topart of the synthetic gene and part of the gene in the host that will beexchanged with the synthetic gene. This will allow for a degree ofhomology between the construct and the host gene so as to allow fortransformation. Once the primers are mixed with the synthetic gene, PCRis conducted to amplify the integration construct.

The integration construct is then ready to transform the host. Theintegration construct is placed in the transformation vector previouslytested. Instead of a vector electroporation can be used to directlytransform the host with the integration construct. The homologous regionsynthetic gene integration construct is then used to transform the yeastor host cells, with the yeast cells then selected for the transformance.The plasmid and host cells are simply incubated together. Followingtransformation of the yeast cells, it is necessary to mate the haploidhost organisms, yeast strains, to produce a diploid yeast straincontaining the synthetic gene.

Preferably, the organisms that are transformed are haploid, so that theyeast or host strains are mated to form a diploid strain. Haploidstrains are used initially because this is comparatively an easier wayto achieve transformation in the final host organism. Use of the haploidstrain approach is advantageous because a homozygous strain is producedonce the two haploid strains are mated. In particular, the gene isinserted into both strands. If a diploid cell is directly transfected,there is a risk that chromosomal segregation will occur, resulting in anumber of generations that are homozygous and do not contain the desiredintegrant. Conversely, if an epigenetic plasmid approach is taken, thenit is preferred to transfect a diploid cell. Once the transformationprocess is complete, stable transformed host organisms are selected for.

An alternative to using an integration construct is to use 1.6 to 2 μmplasmids that have been isolated from Saccharomyces, Kluyvermoces,Torulaspora, or Zygosaccharomyces spp. Advantageously, these epigeneticplasmids are easy to get into the host. Ideally, the vector will resultin multiple insertions into multi copy ribosomal DNA where many stableprotein copies can be produced.

As such, transformation of the host cell generally follows, standardprotocols (as discussed in Higgins and Cregg, 1997)³⁰, whereby theyeast, for example, and construct are mixed and then grown on a plate.

It is preferred for the synthetic gene to be inserted into thechromosome or maintained as an episomal plasmid. It is most preferredfor the synthetic gene to be inserted into the chromosome.

Once the transformed organism is formed, it is necessary to grow thetransformed organism for several generations to ensure that a sufficientpopulation exists to produce the desired amount of polypeptides.Dependent upon whether the gene is inducible or constitutive, once asufficient population is achieved, it may be necessary to induce thegene to produce the desired polypeptide.

The transformed organisms will express the peptide or protein. Theexpression product is either retained within the cytoplasm, attached tothe cell wall, attached to the cell membrane, attached to any cellularorganelle, or excreted into the medium in which the transformed organismis grown. If the protein is retained in or on the yeast or other hostmicroorganism, then the entire host organism can be added to the feed asa supplement. If the protein is secreted, then the medium in which thehost organisms were grown is added directly to the feed as a spray orother liquid, or as a dry application. It is most preferred if the hostretains the expressed peptide or protein.

When the intact yeast or other microorganisms are added as a supplement,they can be added in microencapsulated form, added directly as asuspension of living organisms, or added after collection as a wet ordry cake of microorganisms for supplement. Microencapsulation includesencapsulation in materials that provide better access to thenutrient-providing organism, or that allow for the selection of a sitefor digestion, such as rumen by-pass materials. It should further bepointed out that production of the polypeptide will occur in such amanner that it will either be released into the broth in which thetransformed organism is grown, released into the cytoplasm of theorganism, or attached to the cell wall. As such, once sufficient peptideformation has occurred, the yeast or transformed organism can be addeddirectly to the grain, or the host organisms can be separated from thebroth, and the broth can then be added to the cereal grain.Alternatively, the peptides may be separated from the host organism andthe broth in which it was grown, with the peptides then added directlyto the cereal grain.

The transfected host does not have to be immediately expressed. Instead,the transfected host can be stored in a lyophilized form for a longperiod of time until needed.

The preferred transformed organism is non-toxic, suitable forconsumption by animals, and expresses sufficient amounts of peptide orpolypeptide to positively impact growth and health of animals fed thesupplement. Preferably, the transformed organism is a yeast cell that isauxotrophic, has a synthetic gene located in its nucleus, and ishaploid. The method and resultant products are illustrated in FIGS. 1-7.

EXAMPLES Example 1

It was desired to form a synthetic gene that could be expressed to forma peptide comprised of amino acids desired for animal nutrition. Assuch, the process was initiated by selecting five enzymes produced by,including:

Kpn I-BGGTAC/C, (SEQ ID NO: 1)

Barn H I-BG/GATCC, (SEQ ID NO: 2)

Sal I-BGTCGAC, (SEQ ID NO: 3)

Hind III-BA/AGCTT, (SEQ ID NO: 4)

Not I-BGC/GGCCGC. (SEQ ID NO: 5)

Additionally, four primers were ordered via the internet fromGibco/BRL/Life Technologies, Inc., with primers selected as follows:

Primer 1: is a 5′ GAPDH Sal I

(SEQ ID NO: 6) 5′ AAA AGT CGA CTC GAG TTT ATC ATT ATC AAT    ACT CGC C3′

Primer 2: is a 3′ GAPDH Nsi I—

(SEQ ID NO: 7) 5′ GAT GAT GCA TCA TTT TGT TTA TTT ATG TGT    GTT TAT TCG3′

Primer 3: is a 3′ SYNPEP Not I—

(SEQ ID NO: 8) 5′ AAA AGC GGC CGC CTA TTA CAT TTT AAT CTT    AGT TTT CC3′

Primer 4: is a 5′ SYNPEP Nsi I—

(SEQ ID NO: 9) 5′ AAT GAT GCA TCA TCA TCA TCA TCA CAA GAC    AAA GAT C3′

The primers were suspended in sterile water to form a 100× stock having50 nmoles/microliter (μL). Thus,

$\frac{{31.8\mspace{14mu}{nmoles} \times 1000\mspace{14mu}{\mu L}} = {636\mspace{14mu}{\mu L}}}{50\mspace{14mu}{nmoles}}\mspace{14mu}{of}\mspace{14mu} H_{2}O$As such, 636 μL of water was mixed with Primer 1, Primer 2 had 662 μL ofH2O added thereto, Primer 3 had 566 μL of H₂O added thereto, and Primer4 had 708 μl, of H₂O added thereto. After the primers were suspended inwater, a cocktail for PCR to make a nucleic acid molecule that expressesthe desired peptide was formed. The nucleic acid molecule was known as asynthetic peptide or Synpep. PCR is a standard reaction protocol used inthe industry, which is known as a polymerase chain reaction.

In the cocktail, the following constituents were mixed:

Water 82.2 μL Pfu Buffer (10x) 10.0 μL dNTPs (10 mm) 2.0 μL 5′ SynpepNsi I (50 ng/μL) 5.0 μL 3′ Synpep Not I (50 ng/μL) 5.0 μL PFU Polymerase(2.5 μ/μL) 2.0 μL

PCR Program No. 33 was selected. The Program cycles were as follows:

94° - 45 seconds 94° - 45 seconds 60° - 45 seconds {close oversizebracket} 30 cycles 72° - 30 seconds 72° - 10 minutes

By using the PCR, it was desired to form a nucleic acid molecule ofapproximately 69 base pairs (bp) and to produce an enhanced amount ofproduct. After completion of the PCR, an analytical gel was run to checkfor the product band. A 1% gel was used with 1 kb and 30 by markers. Theanalytical gel did not reveal the desired gene. As this wasinsufficient, the primers were checked to make sure the syntheticpeptide and the primers did not overlap. It was determined that theproduct on the gel was a product of non-specific annealing.

Example 2

The same procedure as disclosed in Example 1 was followed except a 5′patch was ordered. The patch is a 50 by patch and is the followingnucleic acid sequence:

5′ ATC ATC ACA AGA CAA AGA TCA AAA (SEQ ID NO: 10)    TGG TTT GGA AAACTA AGA TTA AAA    TG 3′.

The PCR Program was run again with the cocktail having the followingconstituents:

Water 82.2 μL Pfu Buffer (10x) 10.0 μL dNTPs (10 mm) 2.0 μL 5′ SynpepNsi I (50 ng/μL) 5.0 μL 3′ Synpep Not I (50 ng/μL) 5.0 μL PFU Polymerase(2.5 μ/μL) 2.0 μL

PCR Program No. 33 was selected and slightly revised:

94° - 45 seconds 94° - 45 seconds 60° - 45 seconds {close oversizebracket} 30 cycles (all stages) 72° - 30 seconds 72° - 10 minutes

The PCR product was then run on a 2% gel using the same markers asdisclosed in Example 1. A product band of 68 by was observed, and it wasdetermined that the band was distinct and, therefore, there was no needfor clean-up of the DNA.

The primer was suspended in 528 μL of H₂O.

Example 3

A GAPDH promoter (679 bp) was formed. A PCR cocktail was formed of thefollowing constituents:

Water 76.0 μL Pfu Buffer 10.0 μL dNTPs 2.0 μL Yeast DNA 1.0 μL 5′ GAPDHSal I (100x) 5.0 μL 3′ GAPDH Nsi I (100x) 5.0 μL Pfu Turbo 1.0 μL 100.0μL

PCR Program No. 34 was run:

94° - 1 minute 94° - 1 minute 61° - 1 minute {close oversize bracket} 30cycles (all stages) 72° - 1 minute 72° - 10 minutes

The PCR program was obtained from the suggested cycling parameters forPCR using PFU Turbo. After the PCR Program was finished, the product waschecked on a 2% gel to see if the 679 by band was formed. It wasdetermined that no product was evident, and it was concluded that thePCR parameters needed to be changed, as well as using a differenttemplate.

Example 4

The same procedure as the previous Example 3 was followed, with the PCRcocktail as follows:

Water 75.0 μL Pfu Buffer 10.0 μL dNTPs 2.0 μL Yeast DNA 10.0 μL 5′ GAPDHSal I (100x) 1.0 μL 3′ GAPDH Nsi I (100x) 1.0 μL Pfu Turbo 1.0 μL 100.0μL

PCR Program No. 34 was used, with it slightly revised compared to theprogram listed in Example 3. The Program was as follows:

95° - 5 minutes 94° - 1 minute 52° - 1 minute 72° - 1 minute {closeoversize bracket} 10 cycles (95°, 94°, 52° and 72° stages) 94° - 1minute 61° - 1 minute 72° - 1 minute {close oversize bracket} 20 cycles(94°, 61°, 72° and 72° stages) 72° - 10 minute

No product was located. It was determined that it may be necessary tochange the template.

Example 5

The 68 by product of Example 2 was then mixed in a PCR cocktail so asto, hopefully, produce an 85 by product synthetic nucleic acid moleculefor expressing desired peptides. The cocktail constituents were asfollows:

Water 80.0 μL Pfu Buffer (10x) 10 μL dNTPs (10 mm) 2.0 μL 5′ Synpep NsiI (50 ng/μL) 1.0 μL 3′ Synpep Not I (50 ng/μL) 1.0 μL 68 bp Product(From Example 2) 5.0 μL Pfu Turbo 1.0 μL 100.0 μL

PCR Program No. 33 was conducted.

94° - 1 minute 94° - 1 minute 49° - 1 minute {close oversize bracket} 10cycles (94°, 94°, 49° and 72° stages) 72° - 5 seconds 94° - 1 minute60° - 1 minute {close oversize bracket} 20 cycles (94°, 60°, 72° and 72°stages) 72° - 5 seconds 72° - 10 minutes

After completion of the PCR step, the product was run on a 2% gel todetermine whether the Synpep of 85 by was formed. It was determined thatthe synthetic nucleic acid molecule (Synpep 85 bp) was made.

Example 6

Next, a series of tests were performed to try and form a promoter:

1. PCR w/TAG

template colony template 5 μL Yeast DNA Water 15.0 μL  Water 10.0 μL Mg - buffer 2.5 μL Mg - buffer 2.5 μL MgCl₂ 3.5 μL MgCl₂ 3.5 μL 10^(x)primers 2.5 μL 10^(x) primers 2.5 μL Tag 0.5 μL Tag 0.5 μL dNTPs 1.0 μLdNTPs 1.0 μL

2. PCR w/Pfu Turbo

template colony template 5 μL Yeast DNA Water 25.0 μL  Water 20.0 μL Pfu buffer 2.5 μL Pfu buffer 2.5 μL dNTPs 0.5 μL dNTPs 0.5 μL 10^(x)primers 2.5 μL 10^(x) primers 2.5 μL Pfu Turbo 0.5 μL Pfu Turbo 0.5 μL

PCR Program No. 34 GAPDH (same as before)

95° - 5 minute 94° - 1 minute 52° - 1 minute {close oversize bracket} 10cycles (95°, 94°, 52° and 72° stages) 72° - 1 minute 94° - 1 minute61° - 1 minute 72° - 1 minute {close oversize bracket} 20 cycles (94°,61°, 72° and 72° stages) 72° - 10 minutes

Before products produced in the PCR reactions were run on a 2% agarosegel. It was found that the GAPDH promoter was formed. The promoter was aproduct equal to about 700 bp. The pGAPDH was cloned by PCR from S.C.genemic DNA using GAPDH promoter specific oligos.

Example 7

To cause ligation between the promoter and the synthetic nucleic acidmolecule, it was necessary to extend the Synpep and promoter to causeoverlap from 14 by to 60 bp. This was done to increase the rate ofannealing. The primers for extension were a 3′ link and a 5′ link.

Cocktail:

Water 155.0 μL  Mg - Buffer 20.0 μL  MgCl₂ 16.0 μL  dNTPs 4.0 μL 3′ Link2.0 μL 5′ GAPDH Sal I 1.0 μL Tag 1.0 μL

PCR Program: Robocycler

94° - 10 minutes 94° - 45 seconds 54° - 30 seconds {close oversizebracket} 10 cycles (94°, 94°, 54° and 72° stages) 72° - 1 minute 94° -45 seconds 62° - 30 seconds 72° - 30 seconds {close oversize bracket} 20cycles (94°, 62°, 72° and 72° stages) 72° - 10 minutes

The extended promoter (720 bp) was formed.

Example 8

Cocktail: 200 μL rxn

Water 50.0 μL Pfu buffer 20.0 μL dNTPs  4.0 μL Synpep (85 bp) 20.0 μL 5′Link  2.0 μL 3′ Synpep Not 12.0 μL Pfu Turbo  2.0 μL

Program No. 4: Extend

95° - 5 minutes 94° - 1 minute 59° - 30 seconds {close oversize bracket}30 cycles (all stages) 72° - 45 seconds 72° - 10 minutes

A gel was run and the extended Synpep was formed.

Then PCR ligation of extended promoter and Synpep was conducted.

The PCR Cocktail was a 200 μL rxn, as follows:

Water 74.0 μL Pfu buffer 20.0 μL Template: Promoter (720 bp) 50.0 μLSynpep (110 bp) 50.0 μL dNTPs  4.0 μL Pfu Turbo  2.0 μL 3′ Synpep Not 1 2.0 μL Pfu Turbo  2.0 μL

Program No. 5: Link

94° - 1 minutes 94° - 1 minute 59° - 1 minute {close oversize bracket}20 cycles (all stages) 72° - 45 seconds 72° - 10 minutes

Next, a gel was run to see if a complete gene was formed. A 764 by bandwas observed, which means the extended promoter was formed.

Example 9

The following ratios of amino acids would approximate the neededessential amino acids in corn based diets. Obviously, as the dietingredients change, the limiting amino acids would change. The aminoacids are listed as a ratio, with the need for lysine set to equal 100.This would be congruent with the way amino acids are expressed on anideal amino acid profile.

TABLE 1 Poultry Swine Dairy Beef Lysine 100 100 100 Methionine/Cysteine60 33 20 Threonine 60 0 0 Valine 75 0 0 Isoleucine 60 0 0 Arginine 80 0100 Tryptophan 20 0 0 Histidine 0 0 35

TABLE 2 Ideal Indispensable Amino Acid Profiles (% of Lysine) of a Dietfor Broiler Chicks in Two Age Categories¹ Days Posthatching Amino Acid 0to 21 21-49 Lysine 100 100 Arginine 105 105 Histidine 37 37 Tryptophan16 17 Isoleucine 67 67 Leucine 111 111 Valine 77 77 Phenylalanine +Tyrosine² 105 105 Methionine + Cysteine³ 72 75 Threonine 67 73 Proline33 20 Glycine (or Serine) 67 50 ¹The listed ratios apply to digestibleamino acid concentrations ²A minimum of 50% of the aromatic amino acidsshould be provided as phenylalanine ³A minimum of 50% of thesulfur-containing amino acids should be provided as methionineThis is referenced in Katz, R. S. and D. H. Baker. 1975. Journal ofAnimal Science 41:1355-1361

TABLE 3 Ideal Indispensable Amino Acid Profile (% Lysine) for Pigs inThree Separate Weight Categories Ideal Patterns (%) of Lysine Amino Acid5 to 20 kg 20 to 50 kg 50 to 100 kg Lysine 100 100 200 Threonine 65 6770 Tryptophan 18 19 20 Methionine 30 30 30 Cystine 30 35 40 Methionine +Cystine 60 62 65 Isoleucine 60 60 60 Valine 68 68 68 Leucine 100 100 100Phenylalanine + Tyrosine 95 95 95 Arginine 42 36 30 Histidine 32 32 32This is referenced in Baker, D. H. and T. K. Chung. 1990. FermexTechnical Review 6-4

TABLE 4 Metabolizable Amino Acid Requirements (grams/day) for SteersGaining 2.3 kg per day at Two Body Weights Body Weight Amino Acid 318 kg432 kg Methionine 17.0 17.4 Lysine 55.1 56.2 Histidine 21.3 21.8Phenylalanine 30.6 31.3 Tryptophan 4.3 4.4 Threonine 34.1 34.9 Leucine59.7 61.3 Isoleucine 25.4 26.1 Valine 35.7 36.6 Arginine 57.1 58.3This is referenced in O'Connor et al. 1993. Journal of Animal Science

TABLE 5 Amino Acid Ratio (% of Lysine) Required to Supplement aCorn-Soybean Meal Diet for Swine (24% Soybean Meal) Amino Acid % ofLysine Lysine 100 Isolencine 15 Methionine + Cysteine 100Phenylalanine + Tyrosine 85 Threonine 56 Tryptophane 18 Valine 22

TABLE 6 Amino Acid Ration (% of Arginine) Required to Supplement a Corn-Soybean Meal Diet for Growing Ruminants (13% Crude Protein Diet). AminoAcid % of Arginine Arginine 100 Methionine 9 Lysine 53 Threonine 9Histidine 23

All of these peptide diet requirements can be produced by the presentinvention.

Example 10

The coding region of a synthetic peptide (SYNPEP) was constructed fromsynthetic DNA oligomers using a polymerase chain reaction (PCR). Thiswas to be used to produce a yeast cell that expressed a peptide and wasspecifically designed for feeding poultry 0 to 21 days. The oligimersare listed below in Table 7.

TABLE 7 RESTRICTION PRIMER NAME SITE SEQUENCE 3′ Synpep Not I Not I AAAAGC GGC CGC CTA TTA CAT TTT AAT CTT AGT TTT CC (SEQ ID NO: 8) 5′ Patchnone ATC ATC ACA AGA CAA AGA TCA AAA TCG TTT GGA AAA CTA AGA TTA AAA TG(SEQ ID NO: 10) 5′ Synpep EcoR I EcoR I AAT GGA ATT CAT GCA TCA TCA TCATCA TCA CAA GAC AAA GAT C (SEQ ID NO: 11) 5′ pGAPZbHOint none TAT CCTCAT AAG CAG CAA TCA ATT CCA TCT ATA CTT TAA AAG ATC TTT TTT GTA GAA ATG(SEQ ID NO: 12) 3′ pGAPZbHOint none ACT TTT ATT ACA TAC AAC TTT TTA AACTAA TAT ACA CAT TCC AGC TTG CAA ATT AAA GCC (SEQ ID NO: 13)

The construction of SYNPEP was a two-step procedure. In the first step,the 5′ Patch and 3′ SYNPEP Not I primers (Table 1) were used to generatea 68 bp DNA product. In the second PCR step, the 68 by product was usedas a template, along with the 5′ SYNPEP EcoR 1 and 3′ SYNPEP Not I(Table 1) primers, to produce the complete 85 by SYNPEP coding region.The construct is illustrated in FIG. 1:

The SYNPEP open reading frame was ligated into the EcoRI and Not I sitesof a vector pGAPZb (Invitrogen Corporation), which contained aheterologous glyceraldehyde-3-phosphate dehydrogenase (GAP) promoterfrom Pichia pastoris. The ligation mix was transformed into XL1-bluebacterial cells (Stratagene) and transformants were selected on Zeocincontaining LB plates. Plasmid DNA were extracted from Zeocin resistantcolonies and used as a template for the production of the pGAP-synpepintegration construct. The linear integration fragment was amplified byPCR using the 5′ pGAPZbHOint and 3′ pGAPZbHOint primers (Table 1), whichadd 40 by of HO gene specific DNA to the ends of the fragment to allowfor homologous recombination. The integration construct is illustratedin FIG. 2.

Saccharomyces cerevisiae strain YPH501 (Mat a/α ura 3-52/ura3-52lys2-801 amb/lys2-801 amb ade2-101 och/ade2-101 och trp1-Δ63his3-Δ200/his3-Δ200 leu2-Δ1/leu2-Δ1) was transformed with the linearintegration fragment and plated onto YPD (rich media) containing theantibiotic Zeocin. Those colonies that were Zeocin resistant were grownin YPD broth to an optical density of 0.8 to 1.2 at 600 nm. The cellswere spun down, then resuspended into boiling Laemmli buffer (2% SDS,10% glycerol, 50 mM DTT, 0.002% bromophenol blue, 62.5 mM Tris, pH 6.8)to extract intact proteins. Extracted proteins were applied to anitrocellulose membrane using a dot blot manifold. Dot blots were probedwith rabbit polyclonal IgG anti-His probe primary antibodies at 1:1000dilution (Santa Cruz Biotechnology, #sc-803), followed by goatanti-Rabbit IgG HRP (horseradish peroxidase)-conjugated secondaryantibodies at 1:1000 dilution (Pierce, #31460). The immunoblots werethen developed using the Pierce SuperSignal West Pico ChemiluminescentSubstrate and exposed to Kodak X-OMAT AR film for approximately 5 to 15minutes. It was observed that expression of the Synpep peptide hadoccurred. Positive controls were polyhistidine peptides purchased fromSanta Cruz Biotechnology (His-probe blocking peptide, #sc-803P). Knownquantities of polyhistine peptide were applied to the samenitrocellulose blots to serve as internal standards for quantification.

As such, this Example shows at least one specific construction which canbe used to form an animal feed.

Protein extracts were isolated from yeast cells containing theintegrated gene fragment using hot Laemmli buffer. 100 μl of each samplewere spotted onto a dot blot apparatus on a nitrocellulose membrane.Membranes were probed with rabbit anti-Polyhistidine IgG antibodies,then developed with a goat anti-rabbit IgG-HRP and a chemiluminescentsubstrate, as described in the text. The dot blot showed the expressionof Synpep in yeast. The amount of peptide is estimated 27 μg/ml, basedupon the intensity of the positive controls which contain known amountsof the polyhistidine. Another dot blot showing the expression of Synpepin several yeast strains.

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Thus, there has been shown and described a method for producing agenetically modified yeast organism which fulfills all of the objectsand advantages sought therefor. It will be apparent to those skilled inthe art, however, that many changes, variations, modifications, andother uses and applications for the subject methods and compositions arepossible, and also changes, variations, modifications, and other usesand applications which do not depart from the spirit and scope of theinvention are deemed to be covered by the invention which is limitedonly by the claims which follow.

1. An isolated construct for insertion into a host organism to generatea transgenic organism to be used as a feed supplement for an animal,said construct being prepared by placing a nucleic acid polymer encodinga polypeptide ordinarily exogenous to said organism under control of apromoter, with said construct selected from the group consisting ofplasmids, cosmids, phagemids, and artificial chromosomes, saidpolypeptide comprising the following amino acid: lysine,methionine/cysteine, threonine, valine, isoleucine, histidine; andtryptophan, in a ratio of 6:3:2:1:2:6:1, wherein methionine/cysteine maybe either methionine or cysteine.
 2. The construct of claim 1 whereinsaid construct is a pRS316 plasmid with a GAPDH promoter.
 3. A methodfor producing an improved animal feed for supplementing the diet of ananimal according to the particular nutritional needs of said animal,said method comprising the steps of: (a) ascertaining the nutritionalneeds of the animal by a feed analysis to determine the amino aciddeficiency of the animal fed with a conventional feed, (b) inserting aconstruct into a yeast strain, said construct being prepared by placinga nucleic acid polymer encoding a polypeptide on a vector, wherein saidpolypeptide is ordinarily exogenous to said yeast strain, and the aminoacid composition of said polypeptide includes means for supplementing ananimal diet according to the particular nutritional needs of the animal,(c) allowing expression of the nucleic acid polymer in said construct toproduce a polypeptide, and (d) mixing said polypeptide with saidconventional feed to form the improved animal feed.
 4. A transformedyeast strain to be used as a feed supplement for an animal, saidtransformed yeast strain being prepared by introducing into a hoststrain a nucleic acid polymer encoding a polypeptide under control of apromoter, said nucleic acid polymer, when expressed, producing apolypeptide comprising the following amino acid units: lysine,methionine/cysteine, threonine, valine, isoleucine, arginine, andtryptophan, in a ratio of 100:60:60:75:60:80:20, whereinmethionine/cysteine may be either methionine or cysteine.
 5. Atransformed yeast strain to be used as a feed supplement for an animal,said transformed yeast strain being prepared by introducing into a hoststrain a nucleic acid polymer encoding a polypeptide under control of apromoter, said nucleic acid polymer, when expressed, producing apolypeptide comprising the following amino acid units: lysine,methionine/cysteine, arginine, and histidine, in a ratio of100:20:100:35, wherein methionine/cysteine may be either methionine orcysteine.
 6. A transformed yeast strain to be used as a feed supplementfor an animal, said transformed yeast strain being prepared byintroducing into a host strain a nucleic acid polymer encoding apolypeptide under control of a promoter, said nucleic acid polymer, whenexpressed, producing a polypeptide comprising the following amino acidunits: lysine, arginine, histidine, tryptophan, isoleucine, leucine,valine, phenylalanine/tyrosine, methionine/cysteine, threonine, proline,and glycine/serine, in a ratio of100:105:37:16:67:111:77:105:72:67:33:67, wherein methionine/cysteine maybe either methionine or cysteine with methionine constituting at least50% of the sulfur-containing amino acids in the polypeptide, andphenylalanine/tyrosine may be either phenylalanine or tyrosine withphenylalanine constituting at least 50% of the aromatic amino acids inthe polypeptide, and glycine/serine may be either glycine or serine. 7.A transformed yeast strain to be used as a feed supplement for ananimal, said transformed yeast strain being prepared by introducing intoa host strain a nucleic acid polymer encoding a polypeptide undercontrol of a promoter, said nucleic acid polymer, when expressed,producing a polypeptide comprising the following amino acid units:lysine, arginine, histidine, tryptophan, isoleucine, leucine, valine,phenylalanine/tyrosine, methionine/cysteine, threonine, proline, andglycine/serine, in a ratio of 100:105:37:17:67:111:77:105:75:73:20:50,wherein methionine/cysteine may be either methionine or cysteine withmethionine constituting at least 50% of the sulfur-containing aminoacids in the polypeptide, and phenylalanine/tyrosine may be eitherphenylalanine or tyrosine with phenylalanine constituting at least 50%of the aromatic amino acids in the polypeptide, and glycine/serine maybe either glycine or serine.
 8. A transformed yeast strain to be used asa feed supplement for an animal, said transformed yeast strain beingprepared by introducing into a host strain a nucleic acid polymerencoding a polypeptide under control of a promoter, said nucleic acidpolymer, when expressed, producing a polypeptide comprising thefollowing amino acid units: lysine, isoleucine, methionine/cysteine,phenylalanine/tyrosine, threonine, tryptophan, and valine, in a ratio of100:15:100:85:56:18:22, wherein methionine/cysteine may be eithermethionine or cysteine, and phenylalanine/tyrosine may be eitherphenylalanine or tyrosine.
 9. A transformed yeast strain to be used as afeed supplement for an animal, said transformed yeast strain beingprepared by introducing into a host strain a nucleic acid polymerencoding a polypeptide under control of a promoter, said nucleic acidpolymer, when expressed, producing a polypeptide comprising thefollowing amino acid units: arginine, methionine, lysine, threonine, andhistidine, in a ratio of 100:9:53:9:23.